For many years DNA transfection has been used successfully in a large number of different cells types and tissues1-6. For cells that have been more difficult to transfect with DNA, viral vectors have been used for stable mis-expression7-9. Fewer studies have used RNA for mis-expressing genes10-12, though studies in frog oocytes and early developmental studies have used RNA extensively, delivering it through direct microinjections. One of the reasons that RNA transfection is less widely used than DNA is its limited stability. Whereas viral mis-expression persists, potentially for the life of the cell and in dividing cells through multiple cell divisions, and plasmid transfection can persist through multiple rounds of cell division, RNA is rapidly degraded.
Nevertheless, RNA has several advantages over more traditional plasmid or viral approaches. 1. Since gene expression from an RNA source does not require transcription, the protein product is produced rapidly after the transfection, and since the mRNA has to only gain access to the cytoplasm, rather than the nucleus, typical transfection methods produce result in an extremely high rate of transfection. 2. Plasmid based approaches require that the promoter driving the expression of the gene of interest be active in the cells under study. While some of the more common promoters are active in a wide variety of cell lines, they are more limited in their expression in primary cells and tissues, particularly in postmitotic neurons. 3. The transient nature of the expression could be useful when studying developmental processes that require only a brief “pulse” of a developmental signal or transcription factor13.
Thus, there is a need in the art for improved reagents and methods for RNA based gene misexpression approach.